The overall objective of this project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development. More specifically, how the elaborate networks of blood and lymphatic vessels arise during vertebrate embryogenesis. Both types of vessels are ubiquitous and vital components of vertebrate animals. Blood vessels innervate and supply every tissue and organ with oxygen, nutrients, and cellular and humoral factors. Lymphatic vessels drain fluids and macromolecules from the interstitial spaces of tissues, returning themto the blood circulation. They also play an important role in immune responses. Both types of vessels have also become a subject of great clinical interest in recent years, particularly with the potential shown by antiangiogenic therapies for combating cancer. Many of the recent insights into mechanisms of vessel formation have come from developmental studies including those being performed as part of this project.[unreadable] [unreadable] The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for studying blood vessels. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development- they permit observation of every vessel in the living animal and simple, rapid screening for even subtle vascular-specific mutants.[unreadable] [unreadable] Major aims of the laboratory include:[unreadable] (i) Developing new tools for experimental analysis of vascular development in zebrafish,[unreadable] (ii) Genetic analysis of vascular development,[unreadable] (iii) Analysis of vascular morphogenesis,[unreadable] (iv) Analysis of vascular patterning,[unreadable] (v) Analysis of lymphatic development[unreadable] [unreadable] Developing New Tools for Experimental Analysis of Vascular Development in Zebrafish[unreadable] This has been an important aim of this project. We previously established a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). More recently, we have generated transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize the blood vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish, and recently used these methods to examine blood vessel lumenogenesis and the ontogeny of the lymphatic system. We are continuing to develop new lines useful for vascular imaging in vivo.[unreadable] [unreadable] Genetic Analysis of Vascular Development[unreadable] We use forward-genetic approaches to identify and characterize new zebrafish mutants that affect the formation of the developing vasculature. We are carrying out an ongoing large-scale genetic screen for ENU-induced mutants using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We have screened well over 2000 genomes to date, and identified over 100 new vascular mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. A bulked segregant mapping pipeline is in place to rapidly determine the rough position of newly identified mutants on the zebrafish genetic map, and fine mapping and molecular cloning is in progress for many mutants. We have already positionally cloned the defective genes from a number of vascular patterning mutants, including violet beauregarde (defective in Alk1/acvrl1), y10 (defective in phospholipase C-gamma 1), kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), and etsrp (an ETS-related transcription factor). These mutant screens will continue to yield a rich harvest of novel vascular-specific mutants and bring to light new pathways regulating the formation of the developing vertebrate vasculature.[unreadable] [unreadable] Analysis of Vascular Morphogenesis[unreadable] Classical studies dating back more than 100 years have suggested a model for assembly of endothelial tubes via formation and fusion of vacuoles, but conclusive in vivo evidence for this model has been lacking, primarily due to difficulties associated with imaging the dynamics of sub-cellular endothelial vacuoles deep within living animals. Taking advantage of the favorable optical properties of the fish and novel transgenic lines that we have developed, we have used high-resolution time-lapse two-photon imaging to visualize how the formation and intra- and inter-cellular fusion of endothelial vacuoles drives vascular lumen formation. Ongoing studies are aimed at further characterizing the nature of this process.[unreadable] [unreadable] Analysis of Vascular Patterning[unreadable] We have used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and ongoing studies in the laboratory are aimed at understanding how this pattern arises and the what cues guide vascular network assembly during development. We recently demonstrated that well-known neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning. We showed that Semaphorin-Plexin signaling it is an essential determinant of trunk vessel patterning, uncovering a novel Plexin gene expressed specifically in the vasculature and showing that loss of function of this gene causes dramatic mispatterning of these vessels. Ongoing studies are aimed at further understanding the role of Semaphorin-Plexin signaling in the vasculature and identifying and characterizing additional signals and receptors that function in guidance and patterning during vascular network assembly.[unreadable] [unreadable] Analysis of Lymphatic Development[unreadable] The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes, but progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. We recently showed for the first time that the zebrafish possesses a lymphatic system that shares many of the morphological, molecular, and functional characteristics of the lymphatic vessels found in other vertebrates, providing a superb new model for imaging and studying lymphatic development. Using two-photon time-lapse imaging of transgenic zebrafish, we also traced the migration and lineage of individual cells incorporating into the lymphatic endothelium providing the first conclusive in vivo evidence establishing that early lymphatic endothelial cells are derived from primitive venous blood vessels. We are continuing to examine the assembly and origins of the lymphatic system of the zebrafish in ongoing studies that should provide new insights into the molecular regulation of lymphatic development.